Pathogen-reactive T helper cell analysis in the pig

There is growing interest in studying host-pathogen interactions in human-relevant large animal models such as the pig. Despite the progress in developing immunological reagents for porcine T cell research, there is an urgent need to directly assess pathogen-specific T cells-an extremely rare population of cells, but of upmost importance in orchestrating the host immune response to a given pathogen. Here, we established that the activation marker CD154 (CD40L), known from human and mouse studies, identifies also porcine antigen-reactive CD4(+) T lymphocytes. CD154 expression was upregulated early after antigen encounter and CD4(+)CD154(+) antigen-reactive T cells coexpressed cytokines. Antigen-induced expansion and autologous restimulation enabled a time-and dose-resolved analysis of CD154 regulation and a significantly increased resolution in phenotypic profiling of antigen-responsive cells. CD154 expression identified T cells responding to staphylococcal Enterotoxin B superantigen stimulation as well as T cells responding to the fungus Candida albicans and T cells specific for a highly prevalent intestinal parasite, the nematode Ascaris suum during acute and trickle infection. Antigen-reactive T cells were further detected after immunization of pigs with a single recombinant bacterial antigen of Streptococcus suis only. Thus, our study offers new ways to study antigen-specific T lymphocytes in the pig and their contribution to host-pathogen interactions

Natural heritage inventory of the town of Vail: final report

Prepared for: the town of Vail.March 29, 1994

Breastfeeding, infant formula supplementation, and Autistic Disorder: the results of a parent survey

BACKGROUND: Although Autistic Disorder is associated with several congenital conditions, the cause for most cases is unknown. The present study was undertaken to determine whether breastfeeding or the use of infant formula supplemented with docosahexaenoic acid and arachidonic acid is associated with Autistic Disorder. The hypothesis is that breastfeeding and use of infant formula supplemented with docosahexaenoic acid/arachidonic acid are protective for Autistic Disorder. METHODS: This is a case-control study using data from the Autism Internet Research Survey, an online parental survey conducted from February to April 2005 with results for 861 children with Autistic Disorder and 123 control children. The analyses were performed using logistic regression. RESULTS: Absence of breastfeeding when compared to breastfeeding for more than six months was significantly associated with an increase in the odds of having autistic disorder when all cases were considered (OR 2.48, 95% CI 1.42, 4.35) and after limiting cases to children with regression in development (OR 1.95, 95% CI 1.01, 3.78). Use of infant formula without docosahexaenoic acid and arachidonic acid supplementation versus exclusive breastfeeding was associated with a significant increase in the odds of autistic disorder when all cases were considered (OR 4.41, 95% CI 1.24, 15.7) and after limiting cases to children with regression in development (OR 12.96, 95% CI 1.27, 132). CONCLUSION: The results of this preliminary study indicate that children who were not breastfed or were fed infant formula without docosahexaenoic acid/arachidonic acid supplementation were significantly more likely to have autistic disorder

Femtosecond To Millisecond Dynamics Of Light Induced Allostery In The Avena Sativa LOV Domain

The rational engineering of photosensor proteins underpins the field of optogenetics, in which light is used for spatio-temporal control of cell signalling. Optogenetic elements function by converting electronic excitation of an embedded chromophore into structural changes on the microseconds to seconds timescale, which then modulate the activity of output domains responsible for biological signalling. Using time resolved vibrational spectroscopy coupled with isotope labelling we have mapped the structural evolution of the LOV2 domain of the flavin binding phototropin Avena sativa (AsLOV2) over 10 decades of time, reporting structural dynamics between 100 femtoseconds and one millisecond after optical excitation. The transient vibrational spectra contain contributions from both the flavin chromophore and the surrounding protein matrix. These contributions are resolved and assigned through the study of four different isotopically labelled samples. High signal-to-noise data permit the detailed analysis of kinetics associated with the light activated structural evolution. A pathway for the photocycle consistent with the data is proposed. The earliest events occur in the flavin binding pocket, where a sub-picosecond perturbation of the protein matrix occurs. In this perturbed environment the previously characterised reaction between triplet state isoalloxazine and an adjacent cysteine leads to formation of the adduct state; this step is shown to exhibit dispersive kinetics. This reaction promotes coupling of the optical excitation to successive time-dependent structural changes, initially in the -sheet then -helix regions of the AsLOV2 domain, which ultimately gives rise to J-helix unfolding, yielding the signalling state. This model is tested through point mutagenesis, elucidating in particular the key mediating role played by Q513

Combined Free-running 4D anatomical and flow MRI with native contrast using Synchronization of Neighboring Acquisitions by Physiological Signals (SyNAPS).

BACKGROUND 4D flow MRI often relies on the injection of gadolinium- or iron-oxide-based contrast agents to improve vessel delineation. In this work, a novel technique is developed to acquire and reconstruct 4D flow data with excellent dynamic visualization of blood vessels but without the need for contrast injection. Synchronization of Neighboring Acquisitions by Physiological Signals (SyNAPS) uses Pilot Tone (PT) navigation to retrospectively synchronize the reconstruction of two free-running 3D radial acquisitions, to create co-registered anatomy and flow images. METHODS Thirteen volunteers and two Marfan Syndrome patients were scanned without contrast agent using one free-running fast interrupted steady-state (FISS) sequence and one free-running phase-contrast MRI (PC-MRI) sequence. PT signals spanning the two sequences were recorded for retrospective respiratory motion correction and cardiac binning. The magnitude and phase images reconstructed, respectively, from FISS and PC-MRI, were synchronized to create SyNAPS 4D flow datasets. Conventional 2D flow data were acquired for reference in ascending (AAo) and descending aorta (DAo). The blood-to-myocardium contrast ratio, dynamic vessel area, net volume, and peak flow were used to compare SyNAPS 4D flow with Native 4D flow (without FISS information) and 2D flow. A score of 0-4 was given to each dataset by two blinded experts regarding the feasibility of performing vessel delineation. RESULTS Blood-to-myocardium contrast ratio for SyNAPS 4D flow magnitude images (1.5±0.3) was significantly higher than for Native 4D flow (0.7±0.1, p<0.01), and was comparable to 2D flow (2.3±0.9, p=0.02). Image quality scores of SyNAPS 4D flow from the experts (MP: 1.9±0.3, ET: 2.5±0.5) were overall significantly higher than the scores from Native 4D flow (MP: 1.6±0.6, p=0.03, ET: 0.8±0.4, p<0.01) but still significantly lower than the scores from the reference 2D flow datasets (MP: 2.8±0.4, p<0.01, ET: 3.5±0.7, p<0.01). The Pearson correlation coefficient between the dynamic vessel area measured on SyNAPS 4D flow and that from 2D flow was 0.69±0.24 for the AAo and 0.83±0.10 for the DAo, whereas the Pearson correlation between Native 4D flow and 2D flow measurements was 0.12±0.48 for the AAo and 0.08±0.39 for the DAo. Linear correlations between SyNAPS 4D flow and 2D flow measurements of net volume (r2=0.83) and peak flow (r2=0.87) were larger than the correlations between Native 4D flow and 2D flow measurements of net volume (r2=0.79) and peak flow (r2=0.76). DISCUSSION AND CONCLUSION The feasibility and utility of SyNAPS was demonstrated for joint whole-heart anatomical and flow MRI without requiring ECG gating, respiratory navigators, or contrast agents. Using SyNAPS a high-contrast anatomical imaging sequence can be used to improve 4D flow measurements that often suffer from poor delineation of vessel boundaries in the absence of contrast agents

Variation in LOV Photoreceptor Activation Dynamics Probed by Time-Resolved Infrared Spectroscopy

The light, oxygen, voltage (LOV) domain proteins are blue light photoreceptors that utilize a non-covalently bound flavin mononucleotide (FMN) cofactor as the chromophore. The modular nature of these proteins has led to their wide adoption in the emerging fields of optogenetics and optobiology, where the LOV domain has been fused to a variety of output domains leading to novel light-controlled applications. In the present work, we extend our studies of the sub-picosecond to sub-millisecond transient infrared spectroscopy of the isolated LOV domain AsLOV2 to three full-length photoreceptors in which the LOV domain is fused to an output domain: the LOV-STAS protein, YtvA, the LOV-HTH transcription factor, EL222, and the LOV-histidine kinase, LovK. Despite differences in tertiary structure, the overall pathway leading to cysteine adduct formation from the FMN triplet state is highly conserved, although there are slight variations in rate. However significant differences are observed in the vibrational spectra and kinetics after adduct formation, which are directly linked to the specific output function of the LOV domain. While the rate of adduct formation varies by only 3.6-fold amongst the proteins, the subsequent large-scale structural changes in the full-length LOV photoreceptors occur over the micro- to sub-millisecond timescales and vary by orders of magnitude depending on the different output function of each LOV domain

Clinical relevance of lung transplantation for COVID-19 ARDS: a nationwide study

BACKGROUND: Although the number of lung transplantations (LTx) performed worldwide for COVID-19 induced acute respiratory distress syndrome (ARDS) is still low, there is general agreement that this treatment can save a subgroup of most severly ill patients with irreversible lung damage. However, the true proportion of patients eligible for LTx, the overall outcome and the impact of LTx to the pandemic are unknown. METHODS: A retrospective analysis was performed using a nationwide registry of hospitalised patients with confirmed severe acute respiratory syndrome coronavirus type 2 (SARS-Cov-2) infection admitted between January 1, 2020 and May 30, 2021 in Austria. Patients referred to one of the two Austrian LTx centers were analyzed and grouped into patients accepted and rejected for LTx. Detailed outcome analysis was performed for all patients who received a LTx for post-COVID-19 ARDS and compared to patients who underwent LTx for other indications. RESULTS: Between January 1, 2020 and May 30, 2021, 39.485 patients were hospitalised for COVID-19 in Austria. 2323 required mechanical ventilation, 183 received extra-corporeal membrane oxygenation (ECMO) support. 106 patients with severe COVID-19 ARDS were referred for LTx. Of these, 19 (18%) underwent LTx. 30-day mortality after LTx was 0% for COVID-19 ARDS transplant recipients. With a median follow-up of 134 (47–450) days, 14/19 patients are alive. CONCLUSIONS: Early referral of ECMO patients to a LTx center is pivotal in order to select patients eligible for LTx. Transplantation offers excellent midterm outcomes and should be incorporated in the treatment algorithm of post-COVID-19 ARDS

Risk classification at diagnosis predicts post-HCT outcomes in intermediate-, adverse-risk, and KMT2A-rearranged AML.

Little is known about whether risk classification at diagnosis predicts post-hematopoietic cell transplantation (HCT) outcomes for acute myeloid leukemia (AML) patients. We evaluated 8709 AML patients from the CIBMTR database and, after selection and manual curation of cytogenetics data, 3779 patients in CR1 were included in the final analysis: 2384 with intermediate-risk, 969 with adverse-risk, and 426 with KMT2A-rearranged disease. An adjusted multivariable analysis compared to intermediate-risk patients detected an increased risk of relapse for KMT2A-rearranged and adverse-risk patients (HR 1.27, p = 0.01 and HR 1.71, p < 0.001, respectively). Leukemia-free survival (LFS) was similar for KMT2A and adverse-risk patients (HR 1.26, p = 0.002 and HR 1.47, p < 0.001), as was overall survival (OS) (HR 1.32, p < 0.001 and HR 1.45, p < 0.001). No differences in outcome could be detected when patients were stratified by KMT2A fusion partner. This is the largest study conducted to date on post-HCT outcomes in AML using manually curated cytogenetics for risk stratification. Our work demonstrates that risk classification at diagnosis remains predictive of post-HCT outcomes in AML. It also highlights the critical need to develop novel treatment strategies for patients with KMT2A rearrangements and adverse-risk disease

Excited State Vibrations of Isotopically Labeled FMN Free and Bound to a Light–Oxygen–Voltage (LOV) Protein

Flavoproteins are important blue light sensors in photobiology and play a key role in optogenetics. The characterization of their excited state structure and dynamics is thus an important objective. Here, we present a detailed study of excited state vibrational spectra of flavin mononucleotide (FMN), in solution and bound to the LOV-2 (Light-Oxygen-Voltage) domain of Avena sativa phototropin. Vibrational frequencies are determined for the optically excited singlet state and the reactive triplet state, through resonant ultrafast femtosecond stimulated Raman spectroscopy (FSRS). To assign the observed spectra, vibrational frequencies of the excited states are calculated using density functional theory, and both measurement and theory are applied to four different isotopologues of FMN. Excited state mode assignments are refined in both states, and their sensitivity to deuteration and protein environment are investigated. We show that resonant FSRS provides a useful tool for characterizing photoactive flavoproteins and is able to highlight chromophore localized modes and to record hydrogen/deuterium exchange